Conjugated knottin mini-proteins containing non-natural amino acids

ABSTRACT

Disclosed are knottin peptides containing non-natural amino acids so that they can be formed by chemical conjugation into two or more knottin monomers. The knottin monomers comprise a non-natural amino acid such as an aminooxy residue within the polypeptide sequence. The exemplified dimers were produced by oxime formation between two aldehyde groups present on a polyether linker and an aminooxy functional group that was site-specifically incorporated the knottin. Knottins variants based on EETI (Ecballium elaterium trypsin inhibitor) and AgRP (Agouti-related protein) were engineered to contain integrin-binding loops. These dimers were shown to have increased binding strength to integrins on U87MG tumor cells, achieving significant increases in inhibition of cell adhesion and proliferation. Also disclosed are knottin monomers comprising an aminooxy residue; these may be conjugated to molecules such as doxorubicin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/418,545 filed on Jan. 27, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/435,711 filed on Apr. 14, 2015, which is anational phase filing of International Patent Application No.PCT/US2013/065610 filed on Oct. 18, 2013, which claims priority fromU.S. Provisional Patent Application No. 61/716,363 filed on Oct. 19,2012, the disclosures of which are hereby incorporated by reference intheir entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contract R21 CA143498 awarded by the National Cancer Institute. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of knottin mini-proteins(also known as cystine-knot peptides), and to the field ofchemoselective site-specific oxime conjugation chemistry, in particularincorporating a non-natural amino acid containing an aminooxy sidechain, exemplified for use in knottin mini-proteins.

Related Art

The presently exemplified peptides contain engineered (i.e. artificiallycreated) loops having a high binding affinity for cell surface adhesionreceptors (e.g. integrins) which can mediate binding to theextracellular matrix (ECM). Altered ECM interactions play an importantrole in tumorigenesis.¹ Mediated by various receptors, theseinteractions are often found to enhance tumor proliferation andaggressiveness.^(2,3) Integrins, a family of adhesion receptors, bind tocomponents of ECM providing the anchorage that is necessary for celldivision, migration, and invasion.^(4,5) Several integrins, includingαvβ3, αvβ5, and α5β1 are overexpressed in certain types of cancer andtumor vasculature, and therefore inhibitors of these integrins havegenerated clinical interest.⁶⁻⁹

Many integrin receptors bind to an Arg-Gly-Asp (RGD) peptide motif, andthe residues around it determine specificity and affinity.¹⁰ Using a RGDmotif, different examples of peptides, peptidomimetics, and proteinshave been developed for potential cancer therapy, but peptide scaffoldshave been less explored for such application. Previously, we developednanomolar-affinity αvβ3, αvβ5, and αv5β1 integrin binding peptides byevolving a solvent exposed loop of cystine knot peptides, also known asknottins, using yeast surface display.^(11,12) The presently exemplifiedpeptides may bind all three of the above integrins, or αvβ3 only, orαvβ3/αvβ5 integrins.

Knottins have a disulfide-bonded framework and triple-strandedbeta-sheet fold that often provides remarkable stability in harshconditions, rendering them as promising candidates as pharmacophoricscaffolds for diagnostic and therapeutic applications.^(13,14)Additionally, knottins are attractive for protein engineering, becausethe disulfide-constrained loops tolerate sequence diversity.^(15,16)

Previously, low molecular weight scaffolds, including porphyrins,calixarenes, and carbohydrates have been shown to achieve orders ofmagnitude increase in binding strength through dimerization. However,larger scaffolds such as peptides that target integrins, havedemonstrated only a several fold increase in binding strength withrarely improved therapeutic efficacy.¹⁷⁻²⁵ Thus, there remains a need inthe art for the development of peptides that bind integrins withsignificantly improved binding affinity.

PATENTS AND PUBLICATIONS

Cochran et al. “Engineered Integrin Binding Peptides,” US 2009/0257952,published Oct. 15, 2009, discloses the present engineeredintegrin-binding knottins, namely EETI 2.5F and 2.5D and AgRP 7C.

Namavari, et al., “A Novel Method for Direct Site-specific Radiolabelingof Peptides Using [18F]FDG,” Bioconjug Chem. 2009 March; 20(3): 432-436,discloses a radiolabeled RGD peptide with an aminooxy group.Chemoseletive oxime chemistry was used to provide an easy, one-stepsynthesis of [18F]FDG-RGG and [18F]FDG-cyclo(RGD^(D)YK) as probes forpositron emission tomography of U87MG tumor cells expressing α_(v)β₃integrins. The aminooxy group was part of the C terminal end of thepeptide and coupled to a labeled fluoro-2-deoxyglucose molecule.

Lemieux, et al., “Chemoselective ligation reactions with proteins,oligosaccharides and cells,” TIBTECH, December 1998 (Vol. 16), pp.506-512, discloses that the reaction of aldehydes or ketones withaminooxy group can form oxime bonds used in the synthesis of multiplepeptides. The aminooxy group can be used as a chemoselective couplingpartner to ligate proteins. No specific examples of protein ligationusing this group are given.

Lelievre, et al., “Synthesis of peptide di-aldehyde precursor forstepwise chemoselective ligations via oxime bonds,” Tetrahedron Letters42 (2001), pp 235-238, discloses a two step process for the synthesis ofa tri-branched peptide.

Schaffer et al., “Assembly of high-affinity insulin receptor agonistsand antagonists from peptide building blocks,” Proc. Nat. Acad. Sci.100(8):4435-4439 (2003) discloses chemically linked dimers of peptides(S371, S446) that act as insulin receptor agonists. In one approach, thetwo peptides were prepared recombinantly with glycine/serine linkers; inanother approach, they were chemoselectively ligated. A serine wasattached to an amino group and subsequently oxidized to an aldehydefunction. Triethylene or tetraethylene glycol was functionalized with anoxyamino function at each end and used to chemoselectively ligate twoaldehydes by formation of stable oxime bonds.

Hersel et al., “RGD modified polymers: biomaterial for stimulated celladhesion and beyond,” Biomaterials 24:4385-4415 (2003) disclosesaminooxy-RGD peptide for coupling to another peptide.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

In certain aspects, the present invention concerns engineered knottinproteins (e.g. EETI-II or AgRP) comprising a non-natural amino acid suchas an aminooxy (AO) residue that serves to provide a covalent bond to alinker molecule for creating multimeric (e.g. dimeric) peptides. Theknottins are preferably engineered to contain a binding sequence forspecifically recognizes a target, such as an RGD sequence to bind tointegrins flanked by appropriate residues that facilitate binding. Thelinker molecule is covalently bound to two or more engineered knottins.In certain aspects, the present invention comprises the use of anon-natural amino acid introduced into the knottin for conjugation of anengineered integin binding knottin to another molecule, such as a drug.The exemplified peptide dimers are shown to have increased bindingstrength to integrin receptor targets. At least one aminooxy (AO)residue is in a scaffold portion of a knottin polypeptide chain, and theknottin further contains a binding loop. The binding loop is composed ofan amino acid sequence containing a binding motif, e.g. an RGD motifspecific to bind to at least one of α_(v)β₃ integrin, α_(v)β₅ integrinand α₅β₁ integrin.

In certain aspects of the present invention, the aminooxy (AO) residuehas a side chain that is 2-amino-3-(2-(aminooxy)acetamido)propanoicacid.

In certain aspects of the present invention, the knottin proteins aredimerized through chemical conjugation and a linker molecule. The AOresidue contains a side-chain with an aldehyde group, for attachment tothe linker molecule. Functional groups binding to aldehydes areincorporated into the linker molecules.

In certain aspects of the present invention, the linker molecule isN,N′-((butane-1,4-diylbis(oxy))bis(propane-3,1-diyl))bis(4-formylbenzamide)(FIG. 1B). The length of the linker molecule is selected to giveapproximately a 13 angstrom distance between the two knottin subunitswithin the dimer. This distance helps ensure unhindered interaction ofboth knottin subunits within the dimer to separate integrin receptors.Other distances may be used.

In certain aspects, the present invention relates to a knottin proteincomprising an aminooxy residue conjugated to a small molecule. The smallmolecule contains a ketone or aldehyde group, or has a linker thatcontains a ketone or aldehyde group, that reacts with the AO residue forknottin conjugation. In certain aspects of the present invention, thesmall molecule is a therapeutic anticancer drug, such as doxorubicin, ananthracycline-based chemotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a drawing showing a chemical structures for 4-formyl benzoicacid (4FB) and 4,9-dioxa-1,12-dodecanediamine (1), which reacts to yieldthe dialdehyde linker 2.

FIG. 1B is a schematic representation of oxime ligation of 2 and 3 toyield the dimeric knottin 4.

FIG. 2A are flow cytometry histograms showing integrin (αvβ3, αvβ5, α5,and β1) expression on U87MG cells, a recognized cancer cell model (humanglioblastoma-astrocytoma, epithelial-like cell line), as measured usingintegrin-specific antibodies. Since the α5 subunit can only pair withthe β1 subunit, the α5 histogram represents the amount of α5β1 integrincomplex expressed on the cell surface.

FIG. 2B is a graph showing a competition assay of EETI-based integrinbinding proteins 2.5F dimer and 2.5F monomer to U87MG cells, as measuredby flow cytometry. Alexa488-labeled EETI 2.5F was used as a competitor.Monomeric EETI 2.5F with an introduced aminooxy residue is designated as2.5F_AO. EETI-based peptides containing a scrambled sequence that doesnot bind integrin (FNRDG monomer and FNRDG dimer) are shown as negativecontrols.

FIG. 2C is a graph showing a competition assay of AgRP-based integrinbinding proteins: dimeric 7C and monomeric 7C to U87MG cells, asmeasured by flow cytometry. Alexa488-labeled AgRP 7C was used as acompetitor. Monomeric AgRP 7C with aminooxy residue is designated asMonomeric 7C_AO. EETI-based peptides containing a scrambled sequencethat does not bind integrin (monomeric FNRDG and dimeric FNRDG) areshown as negative controls.

FIG. 3A is a graph measuring comparative inhibition of adhesion of U87MGcells treated with dimeric and monomeric 2.5F to fibronectin, acomponent of ECM. Fibronectin coated plates were incubated with U87MGcells with the indicated concentrations of peptides. Adherent cellsremaining after several wash steps were quantified with crystal violetstaining by absorbance at 600 nm. EETI-based peptides containing ascrambled sequence that does not bind integrin (monomeric FNRDG anddimeric FNRDG) are shown as negative controls. Values were normalized tonegative control containing no competing peptides.

FIG. 3B is a panel of microscope images of the U87MG cells on cultureplates after 24 h treatment with 500 nM of the indicated knottins.

FIG. 4A is a bar graph showing time dependent cytotoxicity of U87MGcells after treatment with the indicated knottin mini-proteins.Cytotoxicity was monitored by incubating U87MG cells on culture plateswith 500 nM of each knottin in 4% FBS media at 37° C. and 5% CO₂ andmeasuring cell viability at 24, 48, and 72 h after the treatment usingthe AlamarBlue indicator dye.

FIG. 4B is a bar graph showing dose dependent cytotoxicity of U87MGcells assessed by treating with different concentrations of knottins for72 h and measuring cell viability using the AlamarBlue indicator dye.

FIG. 5 is a drawing showing the general scheme for the conjugation ofdoxorubicin to the engineered knottin via an oxime bond.

FIG. 6 is a graph showing a competition assay of EETI-based integrinbinding proteins dimeric 2.5F and monomeric 2.5F to U87MG cells comparedto Cilengitide, as measured by flow cytometry. Alexa488-labeled EETI2.5F was used as a competitor.

FIG. 7 is a graph measuring comparative inhibition of adhesion of U87MGcells treated with dimeric and monomeric 2.5F or Cilgenitide tofibronectin, a component of ECM. Fibronectin coated plates wereincubated with U87MG cells with the indicated concentrations ofpeptides. Adherent cells remaining after several wash steps werequantified with crystal violet staining by absorbance at 600 nm. Valueswere normalized to negative control containing no competing peptides.

FIG. 8 is a bar graph showing dose dependent cytotoxicity of U87MG cellsassessed by treating with different concentrations of dimeric ormonomeric 2.5F or Cilgenitide for 72 h and measuring cell viabilityusing the AlamarBlue indicator dye.

FIG. 9 is a drawing showing the chemical structure for 4-formyl benzoicacid (4FB) as shown in FIG. 1A, which was reacted with a differentstarting material to generate a longer cross-linker.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

The term “scaffold portion” or “molecular scaffold” means a polypeptideor portions thereof having a sequence that is used in combination with abinding loop portion and also having a specific three-dimensionalstructure, which presents the binding loop portion for optimal targetbinding. Typically a scaffold portion will be held in place by disulfidelinkages between cysteine residues and will be based on a knottinsequence. The binding loop portion is held in place at terminal ends bythe molecular scaffold portion. The term “molecular scaffold” has anart-recognized meaning (in other contexts), which is also intended here.For example, a review by Skerra, “Engineered protein scaffolds formolecular recognition,” J. Mol. Recognit. 2000; 13:167-187 describes thefollowing scaffolds: single domains of antibodies of the immunoglobulinsuperfamily, protease inhibitors, helix-bundle proteins,disulfide-knotted peptides and lipocalins. Guidance is given for theselection of an appropriate molecular scaffold.

The term “binding loop portion” means a polypeptide having an amino acidsequence of about 9-13 residues. It contains a sequence that isconstructed to bind to a target, thereby referred to an engineered loop.It is exogenous to the scaffold portion, which is derived from aknottin. It will further contain a sequence that is engineered to bindto a target, with high affinity, and that the not a knottin nativetarget. For example, a binding loop portion can be created throughexperimental methods such as directed molecular evolution to bind to aspecific ligand. That is, for example, the sequence contains an RGDsequence or the like, flanked by residues that dictate high affinity andspecificity. It should be noted that these engineered loops areidentified and optimized through combinatorial library screening, orsimply grafted entirely from natural binding sequences present withinknown proteins. The term “specifically recognizes a target” refers tothe presence of the binding loop portion having high affinity forbinding to another molecule, such as binding to integrins, as describedbelow. The present recognition involves binding at nanomolarconcentrations, as exemplified below.

The term “knottin mini-protein”, “knottin peptide”, or “knottin protein”is used as accepted in the art and refers to a member of a family ofsmall proteins, typically 25-50 amino acids in length, that bind tovarious molecular targets, including proteins, sugars and lipids. Theirthree-dimensional structure is minimally defined by a particulararrangement of three disulfide bonds. This characteristic topology formsa molecular knot in which one disulfide bond passes through a macrocycleformed by the other two intrachain disulfide bridges. Although theirsecondary structure content is generally low, knottins share a smalltriple-stranded antiparallel β-sheet, which is stabilized by thedisulfide bond framework.

Specific examples of knottins include the trypsin inhibitor EETI-II fromEcballium elaterium seeds, the neuronal N-type Ca²⁺ channel blockerω-conotoxin from the venom of the predatory cone snail Conus geographus,the Agouti-related protein (See Millhauser et al., “Loops and Links:Structural Insights into the Remarkable Function of the Agouti-RelatedProtein,” Ann. N.Y. Acad. Sci., Jun. 1, 2003; 994(1): 27-35), the omegaagatoxin family, etc.

As will be understood from the description below, the knottins referredto herein are modified to contain a non-natural amino acid and anengineered binding loop, e.g. an integrin-binding loop containing thesequence RGD.

The term “amino acid” includes both naturally occurring and syntheticamino acids and includes both the D and L form of the acids. Morespecifically, amino acids contain up to ten carbon atoms. They maycontain an additional carboxyl group, and heteroatoms such as nitrogenand sulfur. Preferably the amino acids are α and β-amino acids. The termα-amino acid refers to amino acids in which the amino group is attachedto the carbon directly attached to the carboxyl group, which is theα-carbon. The term β-amino acid refers to amino acids in which the aminogroup is attached to a carbon one removed from the carboxyl group, whichis the β-carbon. The amino acids described here are referred to instandard IUPAC single letter nomenclature, with “X” means in the presentsequence listing, AO.

The non-natural amino acid “AO” is defined in the exemplified sequencesas:

The term “aminooxy” is defined below as R′—O—NH2.

The term “EETI” means Protein Data Bank Entry (PDB) 2ETI. Its entry inthe Knottin database is EETI-II. It has the sequence of SEQ ID NO: 1:

(SEQ ID NO: 1) GC PRILMR CKQDSDCLAGCVCGPNGFCG

The term “AgRP” means PDB entry 1HYK. Its entry in the Knottin databaseis SwissProt AGRP_HUMAN, where the full-length sequence of 129 aminoacids may be found. It comprises the sequence beginning at amino acid87. An additional G is added to this construct. It also includes a C105Amutation described in Jackson, et al. 2002 Biochemistry, 41, 7565.

(SEQ ID NO: 10) GCVRLHESCLGQQVPCCDPCATCYC RFFNAF CYCR-KLGTAMNPCSRT

The dashed portion shows a fragment omitted in the “mini” version,below. The bold and underlined portion, from loop 4, is replaced by theengineered binding loop, having RGD sequences described below.

The term includes a “mini”AgRP, in reference to a truncated AgRP thatmeans PDB entry 1MRO. It is also SwissProt AGRP_HUMAN. It has thesequence, similar to that given above,

(SEQ ID NO: 6) GCVRLHESCLGQQVPCCDPAATCYC RFFNAF CYCRwhere the italicized “A” represents an amino acid substitution whicheliminates a free cysteine. The bold and underlined portion, from loop4, is replaced by the below described he RGD sequences in bindingportions.

The term “cystine” refers to a Cys residue in which the sulfur group islinked to another amino acid though a disulfide linkage; the term“cysteine” refers to the —SH (“half cystine”) form of the residue.Binding loop portions may be adjacent to cystines, i.e. there are noother intervening cystines in the primary sequence in the binding loop.

The term “non-natural amino acid” means an amino acid other than the 20proteinogenic alpha-amino acids which in nature are the building blocksof all proteins within humans and other eukaryotes, and which are alsodirectly encoded by the universal genetic code. Such non-natural aminoacids may be obtained commercially, and may be in a protected orunprotected form. See, for examples, the non-natural amino acid productsavailable from HBCChem, Inc. described at http(colon slash slash)hbcchem-inc.com/unnatural_AA.html.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, preferably 80%, more preferably 85%, mostpreferably at least 90% or at least 95% sequence identity to thereference sequence over a specified comparison window, which in thiscase is either the entire peptide, a molecular scaffold portion, or abinding loop portion (˜9-11 residues). Preferably, optimal alignment isconducted using the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol., 48:443 453. An indication that two peptidesequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Another indication for present purposes, that a sequence issubstantially identical to a specific sequence explicitly exemplified isthat the sequence in question will have an integrin binding affinity atleast as high as the reference sequence. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. “Conservativesubstitutions” are well known, and exemplified, e.g., by the PAM 250scoring matrix. Peptides that are “substantially similar” sharesequences as noted above except that residue positions that are notidentical may differ by conservative amino acid changes. As used herein,“sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the NIH Multiplealignment workshop (http colon:slash slashhelixweb.nih.gov/multi-align/). Three-dimensional tools may also be usedfor sequence comparison.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “lower alkyl” means a straight or branched chain hydrocarbonhaving from one to twelve carbon atoms, optionally substituted withsubstituents selected from the group consisting of lower alkyl, loweralkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl,oxo, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyloptionally substituted by alkyl, aminosulfonyl optionally substituted byalkyl, nitro, or lower perfluoroalkyl, multiple degrees of substitutionbeing allowed. Examples of “alkyl” as used herein include, but are notlimited to, n-butyl, n-pentyl, isobutyl, and isopropyl, and the like.

The term “alkylene” means a straight or branched chain divalenthydrocarbon radical having from one to ten carbon atoms, optionallysubstituted with substituents selected from the group which includeslower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl,lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionallysubstituted by alkyl, carboxy, carbamoyl optionally substituted byalkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano,halogen and lower perfluoroalkyl, multiple degrees of substitution beingallowed. Examples of “alkylene” as used herein include, but are notlimited to, methylene, ethylene, n-propylene, n-butylene, and the like.

As used herein, the term “alkyl” refers to a straight or branched chainhydrocarbon having from one to twelve carbon atoms, optionallysubstituted with substituents selected from the group consisting oflower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl,lower alkylsulfonyl, oxo, mercapto, amino optionally substituted byalkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyloptionally substituted by alkyl, nitro, or lower perfluoroalkyl,multiple degrees of substitution being allowed. Examples of “alkyl” asused herein include, but are not limited to, n-butyl, n-pentyl,isobutyl, and isopropyl, and the like.

As used herein, the term “alkoxy” means the group R_(a)O—, where R_(a)is alkyl as defined above and the term “C₁-C₂ alkoxy” means the groupR_(a)O—, where R_(a) is C₁-C₂ alkyl as defined above.

“Haloalkyl” refers to a straight or branched chain hydrocarboncontaining at least 1, and at most 4, carbon atoms substituted with atleast one halogen, halogen being as defined herein. Examples of branchedor straight chained “C₁-C₄ haloalkyl” groups useful in the presentinvention include, but are not limited to, methyl, ethyl, propyl,isopropyl, isobutyl and n-butyl substituted independently with one ormore halogens, e.g., fluoro, chloro, bromo and iodo.

As used herein, the term “haloalkoxy” means the group R_(a)O—, whereR_(a) is haloalkyl as defined above and the term “C₁-C₂ haloalkoxy”means the group R_(a)O—, where R_(a) is C₁-C₂ haloalkyl as definedabove.

The term “aryl” refers to an optionally substituted benzene ring or toan optionally substituted benzene ring system fused to one or moreoptionally substituted benzene rings to form, for example, anthracene,phenanthrene, or napthalene ring systems. Exemplary optionalsubstituents include lower alkyl, lower alkoxy, lower alkylsulfanyl,lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, aminooptionally substituted by alkyl, carboxy, tetrazolyl, carbamoyloptionally substituted by alkyl, aminosulfonyl optionally substituted byalkyl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy,alkoxycarbonyl, nitro, cyano, halogen, lower perfluoroalkyl, heteroaryl,or aryl, multiple degrees of substitution being allowed. Examples of“aryl” groups include, but are not limited to, phenyl, 2-naphthyl,1-naphthyl, biphenyl, as well as substituted derivatives thereof. Asused herein, the term “aralkyl” refers to an aryl or heteroaryl group,as defined herein including both unsubstituted and substituted versionsthereof, attached through a lower alkylene linker, wherein loweralkylene is as defined herein. As used herein, the term “heteroaralkyl”is included within the scope of the term “aralkyl”. The termheteroaralkyl is defined as a heteroaryl group, as defined herein,attached through a lower alkylene linker, lower alkylene is as definedherein. Examples of “aralkyl”, including “heteroaralkyl”, include, butare not limited to, benzyl, phenylpropyl, 2-pyridinylmethyl,4-pyridinylmethyl, 3-isoxazolylmethyl, 5-methyl-3-isoxazolylmethyl, and2-imidazoyly ethyl.

As used herein the term “aralkoxy” means the group R_(b)R_(a)O—, whereR_(a) is alkylene and R_(b) is aryl, both as defined above.

As used herein, the term “alkylsulfanyl” means the group R_(a)S—, whereR_(a) is alkyl as defined above.

As used herein, the term “alkylsulfenyl” means the group R_(a)S(O)—,where R_(a) is alkyl as defined above.

As used herein, the term “alkylsulfonyl” means the group R_(a)SO₂—,where R_(a) is alkyl as defined above.

As used herein, the term “oxo” means the group ═O

As used herein, the term “mercapto” means the group —SH.

As used herein, the term “carboxy” means the group —COOH.

The term “polypeptoid” means a polymer of peptoids, i.e. N-substitutedglycines, as further described in Kirshenbaum et al., “Sequence-specificpolypeptoids: A diverse family of heteropolymers with stable secondarystructure,” Proc. Nat. Acd. Sci. 95(8): 4303-4308 (1998)

General Overview

The present invention relates to compositions and methods for providinga simple and efficient way to increase the binding strength of smallengineered peptides such as knottin mini-proteins. A non-natural aminoacid residue, containing an aminooxy (AO) side-chain, was incorporatedinto the native peptide backbone of two different classes of knottinscaffolds, Ecballium elaterium trypsin inhibitor (EETI) and theAgouti-related protein (AgRP).

As described above, knottin mini-proteins have a characteristicdisulfide-bonded structure, which is illustrated in Gelly et al., “TheKNOTTIN website and database: a new information system dedicated to theknottin scaffold,” Nucleic Acids Research, 2004, Vol. 32, Database issueD156-D159. A triple-stranded β-sheet is present in many knottins. Thespacing between Cys residues is important, as is the molecular topologyand conformation of the RGD-containing integrin binding loop. Theseattributes are critical for high affinity integrin binding. The RGDmimic loop is inserted between knottin Cys residues, but the length ofthe loop must be adjusted for optimal integrin binding depending on thethree-dimensional spacing between those Cys residues. For example, ifthe flanking Cys residues are linked to each other directly, the optimalloop may be shorter than if the flanking Cys residues are linked to Cysresidues separated in primary sequence. Otherwise, particular amino acidsubstitutions can be introduced that constrain a longer RGD-containingloop into an optimal conformation for high affinity integrin binding.

Engineered integrin-binding variants of EETI and AgRP weresite-selectively dimerized through a linker molecule, such as apolyether linker modified to contain two aldehyde groups(N,N′-((butane-1,4-diylbis(oxy))bis(propane-3,1-diyl))bis(4-formylbenzamide)).The conjugation chemistry is based on the chemoselective reaction of AOgroups with aldehydes to form oxime groups. The present invention alsorelates to oxime-based conjugation of knottin mini-proteins to smallmolecules, such as drugs or imaging labels. The linker may be anysuitable polymeric chain, including polyether, alkyl chain, polypeptide,β-peptide, or polypeptoid.

Knottin Mini-Protein Structures

The present knottin mini-proteins comprise a molecular scaffold portion.They are generally held in a rigid three dimensional conformation bydisulfide bonds formed between two cystine residues. Loop portions existbetween the cystines.

Characteristics of a desirable scaffold for protein design andengineering include: 1) high stability in vitro and in vivo, 2) theability to replace amino acid regions of the scaffold with othersequences without disrupting the overall fold, 3) the ability to createmultifunctional or bispecific targeting by engineering separate regionsof the molecule, and 4) a small size to allow for chemical synthesis andincorporation of non-natural amino acids if desired. Scaffolds derivedfrom human proteins are favored for therapeutic applications to reducetoxicity or immunogenicity concerns, but are not always a strictrequirement. Other scaffolds that have been used for protein designinclude fibronectin (Koide et al., 1998), lipocalin (Beste et al.,1999), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Hufton etal., 2000), and tendamistat (McConnell and Hoess, 1995; Li et al.,2003). While these scaffolds have proved to be useful frameworks forprotein engineering, molecular scaffolds such as knottins have distinctadvantages over other molecular scaffolds.

Chemoselective Chemistry

Chemical strategies frequently used for protein or peptide conjugationrely on amine- or thiol-based reactivity endogenous to the 20genetically encoded amino acids. However, it is often difficult toproduce homogeneous and site-selective conjugation products using theseapproaches. Multiple reactive amines (Lysine and N-terminal aminogroups) are present in proteins and peptides, and redox active cysteinescan have unpredictable and undesirable effects on protein folding andstability.

The present invention exploits the chemoselective formation of an oximebond between an aldehyde and an aminooxy functional group, neither ofwhich are found in genetically-encoded amino acids.^(24,27-29) An oximeis formed by condensation of an aminooxy group with either an aldehydeor a ketone, as shown in FIG. 1. The basic scheme for oxime conjugationis as follows:

The above chemistry is exploited in the present invention by providing aknottin with an amino acid having a non-natural side chain. The sidechain will have one of the shown groups, so that another moiety (such asa small molecule, monomer peptide, or linker) can be conjugated by meansof the side chain. In the examples shown in FIGS. 1A and 1B, the peptideis engineered to contain a side chain with aminooxy group, and a linkeris prepared to have ends with aldehydes, specifically benzamides.

In particular, the aminooxy residue exemplified in the present inventionis 2-amino-3-(2-(aminooxy)acetamido)propanoic acid, which has thefollowing structure and is denoted here as residue “AO” in varioussequences described:

As shown above, this residue is incorporated into the knottinpolypeptide chain using standard methods for Fmoc-based solid phasepeptide synthesis.

As can be seen above, the amino acid side chain may be represented as—CH₂—NH—C(═)—CH₂—

. The required aminooxy group is bolded and underlined. Variousmodifications may be employed in the CH₂—NH—C(═O)—CH₂ portion of theside chain, and may be represented using the general formula R′—O—NH₂,where R′ is lower alkyl, alkylene, aryl, alkoxy, haloalkyl, haloalkoxy,aralkoxy, alkylsulfanyl, alkylsulfenyl, or alkylsulfonyl.

The present knottin peptides will generally have about 25-50 amino acidresidues and so may be prepared by solid phase peptide synthesis orequivalent methods. See, e.g. Collins “Water Soluble Solid Phase PeptideSynthesis,” US 2012/0041173, published Feb. 16, 2012.

Methods of synthesis that allow for the incorporation of non-naturalamino acids may be employed, such as described in Shen et al.,“Umpolumng reactivity in amide and peptide synthesis,” Nature 465;1027-1032 (24 Jun. 2010).

Using solid phase peptide synthesis (SPPS), a non-natural amino acidwith either an aminooxy or an aldehyde group can be incorporated atdifferent knottin sequence positions to allow for a variety of linkingsites. Knottin dimers produced through oxime-based chemistry can containlinkers of varying lengths to alter or optimize biological properties.Non-naturals may also be included into the present peptides bymutagenesis, as described, e.g. in Hohsaka et al, “Incorporation ofnon-natural amino acids into proteins,” Curr. Opinion in ChemicalBiology, 6:809-815 (2002).

The present non-natural amino acids will contain a side chain that maybe coupled to a linker; as such, they may contain a side chain that isaminooxy, aldehyde, ketone, alkyne, alkene, aryl halide diene or azide.Guidance for use of such amino acids may be found in U.S. Pat. No.6,858,396 to Dix, entitled “Positively charged non-natural amino acids,methods of making and using thereof in peptides”, issued Feb. 22, 2005;Mao et al. U.S. Pat. No. 8,048,988, issued Nov. 1, 2011, entitled“Compositions containing, methods Involving, and uses of non-naturalamino acids and peptides.” In other embodiments, the present peptidesmay contain side chains that are aminooxy, aldehyde, ketone, alkyne,azide, alkene, aryl halide, or diene. In other embodiments, the presentpeptides may contain side chains that are aminooxy, aldehyde, or ketone.

The side chain used will be selected to be easily reacted with aspecific reactive group contained in a linker. The linker will havefunctional groups on its ends that react with the side chain of eachnon-natural amino acid used.

Furthermore, knottins containing non-natural amino acids according tothe present invention can be conjugated to a therapeutic molecule fordrug delivery. Such small molecules can have an aldehyde or ketonegroup, or a linker that contains an aldehyde or ketone group, to reactwith the aminooxy group present within the knottin.

Knottins Prepared as Dimers

Aminooxy residues were incorporated into two different classes ofknottins for oxime-based dimerization: Ecballium elaterium trypsininhibitor (EETI) and a truncated form of the Agouti-related protein(AgRP). Knottin variants contain an aminooxy residue along with an RGDintegrin binding sequence.

EETI was modified as follows:

(SEQ ID NO: 1) GC  PRILMR  [CKQDSDC*]LAGCV[CGPNGFC**]G.

The bold and underlined portion is replaced with a binding portion. Asillustrated below, an integrin-binding sequence(s) containing the RGDmotif is used instead as this portion. Additional loops, identified hereas loops 2(*) and 3(**), are shown in brackets. These loops are part ofthe scaffold portion can also be varied without affecting bindingefficiency, as is demonstrated below. AS can be seen, they are delimitedby C residues.

EETI 2.5F was evolved from EETI to have specificity for αvβ3, αvβ5, andα5β1 integrin with antibody-like affinity.¹¹ In previous studies, a K15Smutation allowed site-specific attachment of molecular imaging probes tothe knottin N-terminus.

EETI 2.5F: (SEQ ID NO: 2) GCP ₁ R ₂ P ₃ RGDN ₇ P ₈ P ₉ L ₁₀ T₁₁CKQDSDCLAGCVCGPNGFCG

Again, the bolded portion is the binding portion. The followingsequences contain a non-natural AO instead of K at position just carboxyto the binding sequence, which is numbered as 1-11.

EETI 2.5F_AO: (SEQ ID NO: 3) GCPRPRGDNPPLTCXQDSDCLAGCVCGPNGFCG

Another variant was constructed, based on EETI 2.5D, which binds αvβ3and αvβ5 integrin:

EETI 2.5D: (SEQ ID NO: 4) GCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCGEETI 2.5D_AO:  (SEQ ID NO: 5) GCPQGRGDWAPTSCXQDSDCRAGCVCGPNGFCG

Again, the binding portions are the binding portions. AgRP 7C is a38-amino acid knottin peptide evolved from a truncated cystine-knotdomain of AgRP to have antibody-like affinity for αvβ3 integrin.¹² Thisreferenced paper discloses a truncated form of the Agouti-relatedprotein (AgRP), a 4-kDa knottin peptide with four disulfide bonds andfour solvent-exposed loops, used as a scaffold to engineer peptides thatbound to α_(v)β₃ integrins with high affinity and specificity.

Truncated AgRP: GCVRLHESCLGQQVPCCDPAATCY

CYCR (SEQ ID NO: 6) . The bolded portion is replaced by the bindingportions.

AgRP 7C: (SEQ ID NO: 7) GCVRLHESCLGQQVPCCDPAATCYCYGRGDNDLRCYCRAgRP 7C_AO: (SEQ ID NO: 8) GCVRLHESCLGQQVPCCDPAATCYCYGRGDNDLRCYCX

The present peptides can be produced by recombinant DNA methods or bysolid phase peptide synthesis, which has been demonstrated for bothclasses of knottins described here. These peptides can be conjugatedthrough their N-termini to other molecules containing amine-reactivegroups, such as fluorescent dyes, radioisotopes, or small moleculedrugs. Still further, these peptides may be synthesized with non-naturalamino acids that allow for additional crosslinking functionality, suchas alkyne or azide groups used in Huisgen cycloaddition (“click”)chemistry. Crosslinked polystyrene resin containing Rink amide linkers,such as TentaGel S RAM Fmoc resin, (Advanced ChemTech) may be used togive a C-terminal amide upon cleavage. Peptides are cleaved from theresin and side-chains are deprotected with 8% trifluoroacetic acid, 2%triisopropylsilane, 5% dithiothreitol, and the final product isrecovered by ether precipitation. Modified amino acids such as B-alanineare used as the N-terminal amino acid to prevent thiazolidone formationand release of fluorescein during peptide deprotection (Hermanson,1996). Peptides are purified by reverse phase HPLC using an acetonitrilegradient in 0.1% trifluoroacetic acid and a C4 or C18 column (Vydac) andverified using matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF) or electrosprayionization-mass spectrometry (ESI-MS).

The peptides specifically set forth herein may be modified in a numberof ways. For example, the peptides may be further crosslinkedinternally, or may be crosslinked to each other, or the RGD-containingloops may be grafted onto other crosslinked molecular scaffolds. Thereare a number of commercially-available cross-linking reagents forpreparing protein or peptide bioconjugates. Many of these crosslinkersallow dimeric homo- and heteroconjugation of biological moleculesthrough free amine or sulfhydryl groups in protein side chains. Morerecently, other crosslinking methods involving coupling throughcarbohydrate groups with hydrazide moieties have been developed. Thesereagents have offered convenient, facile, crosslinking strategies forresearchers with little or no chemistry experience in preparingbioconjugates.

Dimerization/Multimerization and Linkage

The present knottin monomers are linked to each other and/or to othermolecules by means of the incorporation of a reactive residue within thechain of the monomer at a position determined to be on the surface ofthe folded peptide. The reactive residue contains an aminooxy grouppendant to the peptide chain; the aminooxy group provides a basis for anoxime linkage to another molecule, i.e. a “linking molecule,” groupcontaining an aldehyde or ketone group. The linking molecule has tworeactive aldehyde groups for the preparation of a dimer ofaminooxy-containing knottins; branched linking molecules may be used forhigher order knottin clusters, e.g. trimers, tetramers, pentamers, etc.The linker molecules is chosen to provide a spacer between knottins toallow the engineered binding loop on each knottin to interact with thecognate ligand, e.g. the ability of RGD in a binding loop to bind toαvβ3 integrin on a tumor cell.

As discussed below, the selection of linker spacing and linker siteprovided here allows “clustering” of knottin peptides and a surprisinglevel of binding enhancement of the multimer to the target. A linker asexemplified in FIG. 1A has a diamine portion of 13 Angstroms and 17.3Angstroms in the overall linker shown at compound 2. was used in thepresent examples; linkers are generally chosen to contain flexiblechains of about 20-100 atoms, and may comprise aliphatic and aromaticgroups in a chain.

The AO group of the present knottins may also be used for conjugation toa variety of small molecules, in particular anti-cancer drugs. Asdiscussed below in connection with FIG. 5, an anthracycline antibiotic(doxorubicin) having a —C(═O)CH₂OH structure is coupled to the knottinthrough the keto group. Other molecules, including the many knownanalogs of doxorubicin may be coupled to the present knottins in thisway.

EXAMPLES Example 1: Knottin Monomers

The aminooxy group was substituted for Lys15 in EETI2.5F and Arg38 inAgRP7C since previous studies demonstrated flexibility of thesepositions.^(11,30) See SEQ ID NOs; 3, 5 and 8.

These knottin monomers were prepared by incorporating the aminooxyresidue 2-amino-3-(2-(aminooxy)acetamido)propanoic acid into thepolypeptide using solid phase peptide synthesis with an Fmoc-protectedversion.

Example 2: Dimerization of Knottin Monomers

Because various lengths of diamine chains were readily available fromvendors, the dialdehyde cross-linkers were prepared by conjugating two4-formyl benzoic acid groups (4FB) to a diamine chain (FIG. 1A, 1B).4,9-Dioxa-1,12-dodecanediamine (1) was chosen as the diamine chain toprovide up to an approximately 13 Å distance between two covalentlylinked knottins since a shorter spacer length may hinder the dimericinteraction between the ligand and the receptor.^(21,25) 4FB was coupledto 1 with dicyclocarbodiimide and HOBt in CH₂Cl₂ at 0° C. for 2 h.

The compound 2 was purified with reverse phase HPLC (RP-HPLC) in 76%yield and conjugated to knottins containing an aminooxy residue (3) inphosphate buffer at 25° C. (FIG. 1B). After 1.5 h of incubation, thedimeric knottins (4) were purified with RP-HPLC in 96% yield (Table 1).

TABLE 1 Characteristics of knottin monomers and dimers RP-HPLC^(a)ESI-MS^(b) Peptide R_(t) (min) [M + H]⁺ _(calc) [M + H]⁺ _(exp)Dialdehyde 33.2 469.5 469.3 linker EETI 2.5F_AO 17.6 3365.6 3365.4 EETI2.5F- 23.9 7162.7 7160.4 Dimer AgRP 7C_AO 18.5 4225.8 4226.3 AgRP 7C22.3 8883.1 8883.0 Dimer FNRDG_AO 18.4 3178.3 3177.8 FNRDG-Dimer 23.96789.1 6788.0 ^(a)The gradient used for RP-HPLC is 10-60% solvent B (90%acetonitrile/10% water/0.1% trifluoroacetic acid) over 38 min.^(b)Molecular masses were determined by electrospray ionization massspectrometry (ESI-MS).

Example 3: Competition Binding Assay

Competition binding assays were performed to measure the relativebinding affinities of engineered knottins to U87MG cells expressingαvβ3, αvβ5, and α5β1 integrins (FIG. 2A, 2B, 2C). 2×10⁵ U87MG cells wereincubated at 4° C. for 10 h in integrin binding buffer (20 mM Tris pH7.5, 1 mM MgCl₂, 1 mM MnCl₂, 2 mM CaCl₂, 100 mM NaCl, and 0.1% BSA) andvarying concentrations of the peptides with 0.25 nM of Alexa488-labeledEETI 2.5F as a competitor for dimeric and monomeric EETI 2.5F, and 5 nMof Alexa488-labeled AgRP 7C as a competitor for dimeric and monomericAgRP 7C. Half-maximal inhibitory concentration (IC₅₀) values weredetermined by nonlinear regression analysis using KaleidaGraph (SynergySoftware), and are presented as the average of three separateexperiments.

As shown by the IC₅₀ values, the apparent binding binding affinities ofEETI2.5F and AgRP7C dimers increased by approximately 46- and 4-foldcompared to the monomers, respectively (Table 2).

TABLE 2 U87MG cell binding and adhesion data Ligand Binding IC₅₀ (nM)Adhesion EC₅₀ (nM) EETI 2.5F 1.64 ± 0.03 180 ± 14 EETI 1.57 ± 0.05 N.D.2.5F_AO EETI 2.5F 0.036 ± 0.002  8 ± 2 Dimer AgRP7 C 13 ± 1  N.D. AgRP7C 3.2 ± 0.6 N.D. Dimer FNRDG_AO (—) (—) FNRDG (—) (—) DimerIC₅₀ values from competition binding assays (FIG. 2) and EC₅₀ valuescell adhesion assays (FIG. 3) are summarized. ( - - - ) indicates veryweak to no competition. N.D.=not determinedThe scrambled EETI FNRDG monomer and dimer showed negligible binding,indicating that the binding observed is due to the specific interactionbetween the knottins and the integrins.

Example 4: Inhibition of Integrin-Dependent Cell Adhesion

Since the EETI 2.5F dimer showed remarkable improvement in integrinbinding strength we analyzed its ability to block interaction betweenU87MG cells and fibronectin through a cell adhesion assay (FIG. 3A).Fibronectin is known to interact with αvβ3, αvβ5, and α5β1 integrins,and our previous study showed that EETI 2.5F effectively blocks theseinteractions and detaches cells from fibronectin coated plates.

Varying concentrations of peptides were added to 4×10⁴ cells in 100 μlof IBB, incubated for 1 h at 37° C., 5% CO₂, and gently washed withDulbecco's PBS (DPBS, Invitrogen). Remaining adherent cells wereincubated with 100 μl of 0.2% crystal violet and 10% ethanol for 10 minat room temperature, washed in DPBS and solubilized with 100 μl/well ofa 50:50 mixture of 100 mM sodium phosphate, pH 4.5 and ethanol for 10min. The absorbance at 600 nm was measured using Synergy HT microplatereader (BioTek).

Both EETI 2.5F monomer and dimer were able to inhibit U87MG celladhesion to fibronectin-coated plates in a dose-dependent manner. Asshown by the IC₅₀ values, EETI 2.5F dimer was 100-fold more effective inpreventing U87MG cell adhesion compared to the monomer, while thescrambled FNRDG monomer and dimer controls were not able to inhibit celladhesion (Table 2). When incubated in the presence of 500 nM of EETI2.5F monomer and dimer for 24 h in DMEM media with 4% FBS, U87MG cellsexhibited a rounded morphology (FIG. 3B).

Example 5: Cytotoxicity of EETI 2.5F Monomer and Dimer with U87MG Cells

To demonstrate the cytotoxicity of the EETI 2.5F monomer and dimer,U87MG cells were incubated in medium containing 4% FBS with 1 μM of theknottins on fibronectin coated plates, and the cell viability wasdetermined after 24, 48, and 72 hours of treatment using AlamarBlueindicator reagent (Invitrogen).

Both EETI 2.5F monomer and dimer inhibited U87MG cell proliferation intime-dependent manner, with the dimer exhibiting significantly higherpotency (FIG. 4A). At a concentration of 500 nM, EETI 2.5F dimer induced53%, 68%, and 69% inhibition while EETI 2.5F monomer induced 37%, 44%,and 50% inhibition after 24, 48, and 72 h of treatment, respectively.Dose-dependent inhibition of U87MG proliferation was also observed (FIG.4B). At concentrations of 1000 nM, 100 nM, and 10 nM after 72 hours oftreatment, EETI 2.5F dimer induced 70%, 52%, and 28% inhibition, whilethe EETI 2.5F monomer induced 60%, 30%, and 15% inhibition,respectively. The scrambled EETI FNRDG monomer and dimer did not inducesignificant inhibition, demonstrating that the cytotoxicity was mediatedby binding to the integrins.

Example 6: Conjugation of Drugs for Targeted Delivery

In this example, a knottin containing an aminooxy residue is added to amixture containing a therapeutic molecule, such as doxorubicin. Mixedwith PBS at 25° C., the ketone group on the therapeutic moleculedoxorubicin interacts with the aminooxy group of the non-natural aminoacid on the knottin peptide, producing the conjugate (FIG. 5).

Conjugating a therapeutic small molecule to the knottin peptide providesan effective means for targeted drug delivery. Doxorubicin is used incancer chemotherapy and interacts with DNA. Its ketone group can reactwith the aminooxy group of the knottin peptide monomer. Additionally,the knottins are able to traverse the vasculature, allowing delivery ofthe therapeutic agent to brain tumors.

EETI 2.5D and EETI 2.5F were conjugated with doxorubicin in this manner.The conjugate was purified using reverse phase HPLC with a 0.1% formicacid in acetonitrile gradient over 20 minutes. The conjugate elutes at7.33 minutes (data not shown). The resulting purified conjugate wasfurther verified using electrospray ionization-mass spectrosometry(ESI-MS).

Example 7: Exemplary Peptide Modifications

The above-exemplified knottin sequences may be varied to produce otherknottins for multimerizatioin that have substantial identity to thosedisclosed. The knottin sequences of the present invention, as describedabove, have (i) a scaffold portion, (ii) a binding loop portion, and(iii) an aminooxy (AO) residue or other non-natural amino acid. Thenon-natural amino acid is advantageously placed adjacent to a bindingloop; however, in certain embodiments it may be placed at a terminus.The AO was placed at the C-terminus of AgRP.

The scaffold portion provides the structural framework and is made rigidby disulfide binds between the cystine residues underlined in theexemplary sequence by the engineered EETI-11, 2.5F_AO:

G₁ C₂ P₃R₄P₅R₆G₇D₈N₉P₁₀P₁₁L₁₂T₁₃ C₁₄X₁₅Q₁₆D₁₇S₁₈D₁₉C₂₀L₂₁A₂₂G₂₃C₂₄V₂₅C₂₆G₂₇P₂₈N₂₉G₃₀G₃₁C₃₂G₃₃ (SEQ ID NO:3). One may introduce other residues in the scaffold portion of thepeptide. The scaffold portion is residues G1, C2 and C14 to the carboxyend. The binding loop, as described above, is residues P3-C14. Theunderlined cysteine residues should be maintained in order to preservethe three dimensional structure. The remainder of the scaffold can bevaried by conservative substitutions, e.g 2-3 amino acid substitutions.For example, in GCP-RPRGDNPPLT-CXQDSDCLAGCVCPNGFCG (SEQ ID NO: 3)wherein the italicized L21, A22, V25 and G30 residues may instead beanother amino acid which is A, G, L, S, T, or V. Similarly, the bindingloop may be modified with 1 amino acid substitution, 2 amino acidsubstitutions, or three amino acid substitutions. For example, R4 couldbe H or L. The binding core of RGD (residues 6-8) should not be varied.

As to the binding loop (bolded in SEQ ID NO: 3), the RGD sequence shouldbe invariant, as stated previously. The other positions may besubstituted as taught in Cochran et al. US 20090257952, “EngineeredIntegrin Binding Peptides,” published Oct. 15, 2009. As stated there,the RGD-containing loop may be varied from the specific sequencesdisclosed. For example the loop sequence of EETI 2.5F, shown between Pand C may be varied by 8 of the amino acids, but are invariant as to theRGD sequence. The other residues can be varied to a certain degreewithout affecting binding specificity and potency. For example, if threeof the eleven residues were varied, one would have about 70% identity to2.5F. For guidance in selecting which residues to vary, histograms inFIG. 11 (of the previously cited 20090257952) presents information onlikely residues for each position. For example, in position −3 (thefirst X), one would most likely use a proline residue, based on isolatedmutants that had positive integrin binding. However, His or Leu are alsopossible choices, as shown by their higher incidence in mutants withgood integrin binding properties.

In specific, the present engineered knotttin peptides may be dimers thatcontain integrin binding portions, and the peptide comprising (i) abinding sequence specific to bind to at least one of αvβ5 integrin, αvβ3integrin and α5β1 integrin, and (ii) a knottin protein scaffold, saidbinding sequence being comprised in said knottin protein scaffold andbeing an 11 amino acid engineered integrin binding loop, 11 amino acidslong comprising the sequence RGD, wherein said RGD is in the sequencebetween residues 3 and 7 in an 11 residue binding loop; (iii), and saidknottin protein scaffold, except for the engineered integrin bindingloop, being substantially identical to one of: EETI-II, AgRP, mini-AGRP,agatoxin or miniagatoxin, wherein, when the knottin protein scaffold issubstantially identical to EETI-II, said integrin binding peptide has asequence at least 90% identical having three or fewer amino acidsubstitutions to a peptide.

The present peptides contain at least one non-natural amino acid, inparticular an amino acid residue with a side chain comprising anaminooxy, —N—C(═O)—CH2—O—NH2.

The present knottin may contain a binding loop portion between the 11residues loop, said binding loop being within a scaffold portion,wherein said scaffold portion may have up to two amino acidsubstitutions so as to be at least 90% identical to SEQ ID NO: 3, SEQNO: 5. Residue P1s in the binding loop above, (See SEQ NO: 2) can beselected from the group consisting of A, V, L, P, F, Y, S, H, D, and N;R₂ is selected from the group consisting of G, V, L, P, R, E, and Q; P₃is selected from the group consisting of G, A, and P; N₇ is selectedfrom the group consisting of W and N; P₈ is selected from the groupconsisting of A, P, and S; P₉ is selected from the group consisting of Pand R; L₁₀ is selected from the group consisting of A, V, L, P, S, T,and E; and T₁₁ is selected from the group consisting of G, A, W, S, T,K, and E.

Variants and AgRp Mini Peptide

(SEQ ID NO: 8) GCVRLHESCLGQQVPCCDPAATCYCY ₁ G ₂ RGDN ₆ D ₇ L ₈ R ₉CYCXmay be made, for example as follows, with respect to the binding loop(bolded above):

Y₁ may be F, W or H G₂ may be A, S or V N₆ may be D, Y D₇ may be N or YL₈ may be I, V, or F, and

R₉ may be K or N. The aminooxy (AO) residue is as illustrated above. Inthis embodiment, the AO non-natural amino acid is positioned at thecarboxy terminus of the peptide.

As to the scaffold portion of the peptide (un-bolded) two to three aminoacids may be substituted. This is shown for the following examples:

(SEQ ID NO: 9) GCVRLHE 

 V CLGQQVPCCDPAATCYCY ₁ G ₂ RGDN ₆ D ₇ L ₈ R ₉CYCX, (SEQ ID NO: 11)GCVRLHESCLGQQVPCCDPA 

 GTCYCY ₁ G ₂ RGDN ₆ D ₇ L ₈ R ₉CYCX, (SEQ ID NO: 12) GCVRLHESCLGQQVPCC 

 E PAATCYCY ₁ G ₂ RGDN ₆ D ₇ L ₈ R ₉CYCX,and so forth.

Variations of EETI-II

As further examples, one may vary the location of the non-natural aminoacid. EETI-II 2.5F_AO has the AO (shown as “X”) in place of the lysineat position 15: GC

QDSDCLAGCVCGPNGFCG (SEQ ID NO: 3). In other words, As before, thebinding loop is bolded. EETI 2.5F_AO may be shown by residue number asfollows:

(SEQ ID NO: 3) G₁C₂ P ₃ R ₄ P ₅ R ₆ G ₇ D ₈ N ₉ P ₁₀ P ₁₁ L ₁₂ T ₁₃C₁₄ X₁₅Q₁₆D₁₇S₁₈D₁₉C₂₀L₂₁A₂₂G₂₃C₂₄ V₂₅C₂₆G₂₇P₂₈N₂₉G₃₀F₃₁C₃₂G₃₃.

EETI 2.5F_AO-2 is GC

CQDSDCLAGCVCGPNGFCGX (SEQ ID NO: 13). This exemplified addition of theAO (shown as “X”) to the carboxyl terminal end, i.e. following residue33.

Using the above numbering, one may see that the AO residue or othernon-natural amino acid may be used, for example in place of any one ofresidues AO15-G23, residues G27-F31, or other loops. Another variant wasconstructed, based on EETI 2.5D, which binds αvβ3 and αvβ5 integrin:

EETI 2.5D (SEQ ID NO: 4) GC PQGRGDWAPTS CKQDSDCRAGCVCGPNGFCGEETI 2.5D_AO (SEQ ID NO: 5) GC PQGRGDWAPTS CXQDSDCRAGCVCGPNGFCG,

X=AO.

EETI 2.5D_AO has the same numbering as the EETI2.5F_AO, although thesequence is different; the AO residue is at position 15, as in EETI2.5F-AO. Variations in sequences may be made as described in connectionwith sequence EETI 2.5F.

Example 8: Comparison of EETI 2.5F Knottin Monomer and Dimer toCilengitide

As is known, Cilengitide is a molecule designed and synthesized at theTechnical University Munich in collaboration with Merck KGaA inDarmstadt. It is based on the cyclic peptide cyclo(-RGDfV-), which isselective for αv integrins, which are important in angiogenesis (formingnew blood vessels). Hence, it is under investigation for the treatmentof glioblastoma by inhibiting angiogenesis. The European MedicinesAgency has granted cilengitide orphan drug status.

Relative U87MG binding affinity, and inhibition of U87MG cell adhesionand proliferation was measured for the EETI 2.5F dimer, EETI 2.5Fmonomer, and Cilengitide as described in Examples 3, 4, and 5. Therelative binding affinity (IC₅₀) of Cilengitide to U87MG cells (using0.25 nM of Alexa488-labeled EETI 2.5F as the competitor) was 41±2 nMcompared to 1.64±0.03 nM for the EETI 2.5F monomer and 0.036±0.002 nMfor the EETI 2.5F dimer (FIG. 6). Unlike the EETI 2.5F dimer and EETI2.5F monomer, Cilengitide does not inhibit U87MG cell adhesion tofibronectin at the highest concentration tested (FIG. 7). Remarkably,the EETI 2.5F dimer is 100-fold more potent at inhibiting cellproliferation compared to Cilengitide (FIG. 8).

Example 9: Pharmaceutical Compositions

The present engineered knottins may be used for imaging or fortherapeutic purposes, as demonstrated above. The present peptides mayalso be formulated as pharmaceutical compositions for use in vivo inhumans. Suitable formulations may be derived by reference to U.S. Pat.No. 7,262,165, entitled “Aqueous preparation containing oligopeptidesand etherified cyclodextrin,” issued Aug. 28, 2007. Briefly, asdescribed there, the knottin to be used will be soluble in water or asuitable buffer such as physiological saline. Based on this solubility,a concentration of knottin peptide in the solution of the pharmaceuticalcomposition may be determined. For example, if the peptide has asaturation solubility in physiological saline solution of about 19 mg/mland it can therefore, for therapeutic use, be safely administeredparenterally in a concentration of 15 mg/ml dissolved in physiologicalsaline solution. If, for example, a dose of 1500 mg is necessary fortherapy with the peptide, a volume to be administered of 100 ml arises.Volumes in this order of magnitude can no longer simply be injected andmust be infused, which is disadvantageous. Pharmaceutically acceptableingredients may be added to increase solubility or tolerance forinjection. The present peptides may also be formulated in enteric form(see e.g. U.S. Pat. No. 5,350,741). Other formulations which may beemployed are in Remington: The Science and Practice of Pharmacy, 19thEdition (1995) and/or Handbook of pharmaceutical granulation technology,chapter 7, “Drugs and the pharmaceutical sciences”, vol. 81, 1997. Infurther embodiments of the present compositions, carriers are selectedfrom hydrophilic binders, water-soluble diluents, surfactants,detergents, lubricants, disintegrants, antioxidants, non water-solublediluents and/or other fillers known to the skilled person. In aparticular embodiment the one or more carriers comprises at least ahydrophilic binder and a water-soluble diluent. Lyophilzed formulationsmay be prepared as described in U.S. Pat. No. 7,265,092, “Pharmaceuticalcompositions,” issued Sep. 4, 2007, to Li. as described there, variousexcipients and anti-aggregants may be used.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly, intraperitoneal, intravenously,and in the case of the present invention via intra-tumor injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid (e.g., dried or lyophilized) formssuitable for reconstitution into solution or suspension in liquid priorto injection, or as emulsions. Generally, suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, minor amounts of non-toxic auxiliary substances can beemployed, such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, tonicifiers and the like including, for example,sodium acetate, sorbitan monolaurate, triethanolamine oleate,cyclodextrins, etc. Dosage forms for intravenous (IV) administrationgenerally comprise an active peptide agent incorporated into a sterilesolution of simple chemicals such as sugars, amino acids orelectrolytes, which can be easily carried by the circulatory system andassimilated. Such solutions are typically prepared with saline orbuffer. The pH of such IV fluids may vary, and will typically be from3.5 to 8.0, as known in the art.

Example 10: Cell Inhibition of Proliferation with Dimer Knottins

Dimers of the present knottins with AO groups incorporated at differentpositions were prepared and conjugated with different linkages. Therewere tested for activity against carcinoma lines. Results are shownbelow in Table 3. As identified there, Dimer 1 is EETI 2.5F where the AOgroup was incorporated in place of Lys15, conjugated through the 17.3Angstrom linker shown in FIG. 1B; Dimer 2 is EETI 2.5F, where the AOgroup was incorporated in place of Lys15, conjugated though the 68.5Angstrom linker shown in FIG. 9. Dimer 3 is EETI 2.5F, where the AOgroup was incorporated at the C-terminus following residue 33,conjugated through a 17.3 Angstrom linker shown in FIG. 1B; Dimer 4 isEETI 2.5F, where the AO group was incorporated at the C-terminusfollowing residue 33, conjugated through the 68.5 Angstrom linker shownin FIG. 9. It was observed that the position of the AO residue had astrong effect on binding activity. Peptide dimers with AO at position at15 exhibited stronger binding than peptides with AO at the C-terminus.Also, it was observed that the length of the linkage had only a minoreffect on binding. Binding, of course, it correlated with the activityin proliferation and apoptosis.

TABLE 3 Apoptosis Proliferation (Caspase-3) Cell line: MDA231 (Humanbreast adenocarcinoma) Cilengitide Weak inhibition Moderate induction2.5F Monomer Weak inhibition Moderate induction 2.5F Dimer1 Stronginhibition Strong induction 2.5F Dimer2 Strong inhibition N/A 2.5FDimer3 Moderate N/A inhibition 2.5F Dimer4 Moderate N/A inhibition Cellline: SKOV 3 (Ovarian adenocarcinoma) Cilengitide N/A N/A 2.5F MonomerN/A N/A 2.5F Dimer1 Strong inhibition N/A 2.5F Dimer2 Strong inhibitionN/A 2.5F Dimer3 N/A N/A 2.5F Dimer4 N/A N/AThe results above indicate that EETI 2.5F Dimers 1 and 2 more stronglyinhibited tumor cell proliferation compared to Dimers 3 and 4,demonstrating that the location of the AO group within the knottinmattered (position 15 versus C-terminus), but the crosslinker length didnot (17.3 versus 68.5 angstroms). All dimers, however, were more potentat inhibiting tumor cell proliferation compared to EETI 2.5F monomer andCilengitide.

Example 11: Additional Linkages

As described above, the present dimers are constructed of two engineeredknottin peptides (e.g. EET-II and AgRP), where the peptides contain abinding loop, and, in the scaffold portion, at least one non-naturalamino residue. The non-natural amino residue has a side chain that canbond to a linkage molecule. By example, the non-natural amino acidcontains an aminooxy functional group. The linker molecule containsfunctional groups in their terminal portions. Each linker molecule canbond to two different engineered peptides. For example, the linkermolecules are terminated with aldehyde groups. Alternative, the linkermolecules can carry more than two functional groups, to form multimersof 3, 4, 5, or more, engineered knottins.

FIG. 9 illustrates linkage molecules having terminal aldehyde groups(4-formylbenzamide) and an alkoxy chain linking the aldehylde-bearinggroups. In the compound in FIG. 9,O,O′-Bis(2-aminoethyl)octadecaethylene glycol was reacted with 4-Formylbenzoic acid (4FB) (similarly to the method of Example 2) to generatethe crosslinker:N,N′-(3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57-nonadecaoxanonapentacontane-1,59-diyl)bis(4-formylbenzamide).As can be appreciated, the various ethylene glycol monomers can bevaried; instead of 18, one could use 2, as in FIG. 1, or any number inbetween. One could use more than 18, e.g. as between 18 and 40 ethyleneglycol monomers.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification,including the below cited references are indicative of levels of thoseskilled in the art to which the patent pertains and are intended toconvey details of the invention which may not be explicitly set out butwhich would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference, asneeded for the purpose of describing and enabling the methods andmaterials.

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1. A knottin peptide comprising a scaffold portion having a sequenceessentially identical to a sequence of EETI-II (SEQ ID NO: 1) or ascaffold portion essentially identical to AgRP (SEQ ID NO: 10); anexogenous binding loop portion within the scaffold portion said bindingloop having a sequence which specifically recognizes a target; and anon-natural amino acid comprised in the scaffold portion.
 2. The knottinpeptide of claim 1 wherein the non-natural amino acid contains a sidechain that is either aminooxy, aldehyde, ketone, alkyne, alkene, arylhalide diene or azide.
 3. The knottin peptide of claim 1 furthercomprising a linker molecule and wherein said knottin peptide is part ofa dimer wherein monomer knottin peptides are linked by the linkermolecule.
 4. The knottin peptide of claim 3 wherein monomers have asequence at least 90% or at least 95% identical to a peptide of SEQ IDNO: 3 (EETI 2.5F_AO); SEQ NO: 13 (2.5F-AO-2); SEQ ID NO: 5 (EETI2.5D_AO); or SEQ ID NO: 8 (AgRP 7C_AO).
 5. The knottin peptide of claim1 wherein the scaffold portion is from EETI-II; the non-natural aminoacid has an aminooxy residue; and said aminooxy residue is at a positionthat is between residues 15 and 23, or else at a position betweenresidues 27-31, inclusive.
 6. The knottin peptide of claim 1 wherein theknottin peptide comprises a scaffold portion from AgRP and thenon-natural amino acid has an aminooxy residue between residues 35 and38, inclusive.
 7. The knottin peptide of claim 1 wherein the non-naturalamino acid is a single residue that has a side chain aminooxy residuethat is 2-amino-3-(2-(aminooxy)acetamido)propanoic acid.
 8. The knottinpeptide of claim 1 wherein the binding loop portion contains thesequence RGD and is between two adjacent cystines in the peptide.
 9. Acomposition as defined in claim 7 wherein said knottin peptide and asecond knottin peptide are bound to each other by a linker molecule. 10.The composition of claim 9 wherein the linker molecule is conjugated toan aminooxy residue incorporated within each knottin peptide.
 11. Thedimer of claim 10 formed with a linker molecule which is a polyether,alkyl chain, polypeptide, β-peptide, or peptoid.
 12. A knottin peptidecomprising an aminooxy residue, wherein said residue is conjugated to asmall molecule, or is conjugated to a molecular linker.
 13. The knottinpeptide of claim 12 having a sequence at least 90% identical or at least95% identical to a peptide listed as SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO:
 8. 14. The knottin peptide of claim 12 wherein the small moleculeis doxorubicin. 15.-27. (canceled)
 28. A method of making a knottin-drugconjugate, comprising: conjugating a small molecule drug to a knottinpeptide, wherein the knottin peptide comprises a non-natural amino acidcomprising a functional group selected from the group consisting of:aldehyde, ketone, alkyne, alkene, aryl halide, diene, azide, andaminooxy, wherein the conjugating comprises conjugating the smallmolecule drug to the non-natural amino acid via a linker molecule.